title: A homogeneous time-resolved fluorescence assay to identify inhibitors of HIV-1 fusion.
abstract: The human immunodeficiency virus type 1 (HIV-1) initiates infection through sequential interactions with CD4 and chemokine coreceptors unmasking the gp41 subunit of the viral envelope protein. Consequently, the N-terminal heptad repeats of gp41 form a trimeric coiled-coil groove in which the C-terminal heptad repeats collapse, generating a stable six-helix bundle. This brings the viral and cell membrane in close proximity enabling fusion and the release of viral genome in the cytosol of the host cell. In this chapter, we describe a homogeneous time-resolved fluorescence assay to identify inhibitors of HIV-1 fusion, based on the ability of soluble peptides, derived from the N- and C-terminal domains of gp41, to form a stable six-helix bundle in vitro. Labeling of the peptides with allophycocyanin and the lanthanide europium results in a Föster resonance energy transfer (FRET) signal upon formation of the six-helix bundle. Compounds interfering with the six-helix bundle formation inhibit the HIV-1 fusion process and suppress the FRET signal.
authors.affiliation: Janssen Infectious Diseases-Diagnostics BVBA, Johnson & Johnson Corporation, Beerse, Belgium.
authors.forename: Liesbet
authors.initials: L
authors.lastname: Smeulders
authors.forename: Lieve
authors.initials: L
authors.lastname: Bunkens
authors.forename: Inge
authors.initials: I
authors.lastname: Vereycken
authors.forename: Koen
authors.initials: K
authors.lastname: Van Acker
authors.forename: Pascale
authors.initials: P
authors.lastname: Holemans
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Gustin
authors.forename: Marnix
authors.initials: M
authors.lastname: Van Loock
authors.forename: Géry
authors.initials: G
authors.lastname: Dams
mesh_terms: D015200:Fluoroimmunoassay / Q000379:methods; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000378:metabolism; D023581:HIV Fusion Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects
chemical_list: D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D010455:Peptides